The Polymerase Chain Reaction (PCR), is a recently developed technique which has had a significant impact in many areas of science. PCR is a rapid and simple method for specifically amplifying a target DNA sequence in an exponential manner. (Saiki et al. (1985) Science 230:1350; Mullis and Faloona (1987) Methods Enzymol. 155:335). Briefly, the method consists of synthesizing a set of primers that have nucleotide sequences complementary to the DNA that flanks the target sequence. The primers are then mixed with a solution of the target DNA, a thermostable DNA polymerase and all four deoxynucleotide triphosphates (dATP, dTTP, dCTP and dGTP). The solution is then heated to a temperature sufficient to separate the complementary strands of DNA (approximately 95.degree. C.) and then cooled to a temperature sufficient to allow the primers to bind to the flanking sequences. The reaction mixture is then heated again (to approximately 72.degree. C.) to allow the DNA synthesis to proceed. After a short period of time, the temperature of the reaction mixture is once again raised to a temperature sufficient to separate the newly formed double-stranded DNA, thus completing the first cycle of PCR. The reaction mixture is then cooled and the cycle is repeated. Thus, PCR consists of repetitive cycles of DNA melting, annealing and synthesis. Twenty replication cycles can yield up to a million fold amplification of the target DNA sequence. The ability to amplify a single DNA molecule by PCR has applications in environmental and food microbiology (Wemars et al. (1991) Appl. Env. Microbiol. 57:1914-1919; Hill and Keasler (1991) Int. J. Food Microbiol. 12:67-75), clinical microbiology (Wages et al. (1991) J. Med. Virol. 33:58-63; Sacramento et al. (1991) Mol. Cell Probes 5:229-240; Laure et al. (1988) Lancet 2:538), oncology (Kumar and Barbacid (1988) Oncogene 3:647-651; McCormick (1989) Cancer Cells 1:56-61; Crescenzi et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:4869), genetic disease prognosis (Handyside et al (1990) Nature 344:768-770), blood banking (Jackson (1990) Transfusion 30:51-57) and forensics (Higuchi et al. (1988) Nature (London) 332:543).
The availability of thermostable DNA polymerases such as Taq DNA polymerase has both simplified and improved PCR. Originally only heat-sensitive polymerases, such as E. coli DNA polymerase were available for use in PCR. Heat-sensitive polymerases, however, are destroyed at the temperatures required to melt double-stranded DNA and additional polymerase has to be added after each PCR cycle. Taq DNA polymerase, isolated from the thermophilic bacterium Thermus aquaticus, is stable up to 95.degree. C. and its use in PCR has eliminated the necessity of repetitive addition of temperature sensitive polymerases after each thermal cycle. Additionally, because Taq polymerase can be used at higher temperatures it has improved the specificity and sensitivity of PCR. The reason for the improved specificity is that at higher temperatures the binding of primers to sites other that the desired ones (referred to as mispriming) is significantly reduced.
Since its discovery, the Polymerase Chain Reaction has been modified for various applications, such as in situ PCR, in which the detection limit of traditional in situ hybridization has been pushed to the single copy level (Haase et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:4971-4975), and reverse transcriptase PCR (RT-PCR), wherein an RNA sequence is converted to its copy DNA (cDNA) by reverse transcriptase (RT) before being amplified by PCR, making RNA a substrate for PCR (Kawasaki (1991) Amplification of RNA in PCR Protocols, A Guide to Methods and Applications, Innis et al., Eds. Academic Press Inc., San Diego, Calif., 21-27). Mesophilic viral reverse transcriptases, however, are often unable to synthesize full-length cDNA molecules because they cannot "read through" stable secondary structures of RNA molecules. This limitation has recently been overcome by use of a polymerase isolated from Thermus thermophilus (Tth polymerase). Tth polymerase is a thermostable polymerase that can function as both reverse transcriptase and DNA polymerase (Myers and Gelfand (1991) Biochemistry 30:7661-7666). The reverse transcription performed at an elevated temperature using Tth polymerase eliminates secondary structures of template RNA, making it possible for the synthesis of full-length cDNA.
Although significant progress has been made in PCR technology, the amplification of nontarget oligonucleotides due to side-reactions, such as mispriming of background DNA and/or primer oligomerization still presents a significant problem. This is especially true in diagnostic applications in which PCR is carried out in a milieu containing background DNA while the target DNA may be present in a single copy (Chou et al. (1992) Nucleic Acid Res. 20:1717-1723). The generation of nonspecifically amplified products has been attributed to polymerase activity at ambient temperature that extends nonspecifically annealed primers. (Chou et al. (1992) Nucleic Acid Res. 20:1717-1723, Li et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:4580). Accordingly, the inhibition of polymerase activity at ambient temperature is important to control the generation of non-specific products.
Two methods have been reported which minimize these side reactions. In the first method, termed "manual hot start" PCR, a component critical to polymerase activity (e.g. divalent ions and/or the polymerase itself) is not added to the reaction mixture until the temperature of the mixture is high enough to prevent nonspecific primer annealing. (Chou et al. (1992) Nucleic Acid Res. 20:1717-1723; D'Aquila et al. (1991) Nucleic Acid Res. 19:3749). Thus, all of the reagents are heated to 72.degree. C. before adding the final reagent, usually the polymerase. In wax-mediated "hot start" PCR, a component(s) crucial to polymerase activity is physically separated from the rest of the reaction mixture at low temperature by a wax layer which melts upon heating in the first cycle. (Chou et al. (1992) Nucleic Acids Res. 20:1717; Horton et al. (1994) BioTechniques 16:42). "Hot start" PCR has certain drawbacks; the requirement of reopening of tubes before initiating thermocycling increases crossover contamination and repetitive pipetting makes it tedious in handling a large number of samples. A reagent that could be placed directly in the reaction mixture with all other reaction components and inhibit the polymerase at ambient temperature would be useful to overcome limitations associated with "hot start" PCR. Although this method does increase specificity, thereby reducing side products, the method is inconvenient for dealing with a large number of samples, the reaction mixture can become more easily contaminated, and the method is error-prone.
In the second method, termed "in situ hot start," a reagent that binds and inhibits the polymerase at low temperature, but not at high temperature, (e.g. a neutralizing antibody to Taq polymerase (TaqStart) or an oligonucleotide aptamer) is added to the complete reaction mixture. (Birch et al. (1996) Nature 381:445, Dang and Jayasena (1996) J. Mol. Biol. 264:268; Kellogg et al. (1994) Bio Techniques 16:1134-1137). This antibody inhibits the polymerase activity at ambient temperature, but is inactivated by heat denaturation once the reaction is thermocycled, rendering the polymerase active. The drawback of this approach to reducing side products is that the anti-Taq antibody should be stored at -20.degree. C. until use, which means that detection kits should be packaged and shipped under controlled environment adding to their cost. In addition, a significant amount of antibody (.about.1 .mu.g of antibody/5 U of Taq polymerase), diluted in a vendor specified buffer, is needed for a single PCR.
The development of high affinity nucleic acid ligands capable of inhibiting the thermostable Taq and Tth polymerases would obviate the need for the "hot start" method and would overcome the limitations associated with the second method. Nucleic acid inhibitors can be developed that are extremely specific and have high affinity. Since nucleic acids are more stable than proteins at ambient temperature, the shipping and packaging problems associated with using antibodies can be overcome. Additionally, nucleic acids, like antibodies can be identified that will lose their affinity for the polymerase at higher temperatures, allowing the polymerase to be activated when desired. The potential for mispriming mediated by nucleic acid based inhibitors themselves functioning as primers (in addition to the specific primers used in the reaction) in PCR can be eliminated by capping their 3' ends.
X-ray crystal structures of several DNA polymerases have indicated that they fold into similar three dimensional structures. (For a review, see Joyce and Steitz (1994) Annu. Rev. Biochem. 63:777). The C-terminal domain responsible for polymerization is organized into three sub-domains representing "palm," "fingers" and "thumb," anatomically analogous to a right hand. Tth polymerase and Taq polymerase are 93% similar and 88% identical at the amino acid sequence level (Abramson (1995) in PCR Strategies (Academic Press, New York). Both are devoid of 3'.fwdarw.5' exonuclease activity, but contain 5'.fwdarw.3' exonuclease activity (Abramson (1995) in PCR Strategies (Academic Press, New York); Tindall and Kunkel (1988) Biochemistry 27:6008). Thus, nucleic acid ligand inhibitors might be expected to behave similarly toward both of these enzymes, as well as, other thermostable polymerases. This would make possible the use of a single inhibitor for a number of thermostable enzymes.
RNA sequences are converted to cDNA by reverse transcription before being amplified by PCR. Initially, this was achieved in two steps using two different enzymes: a reverse transcriptase and a thermostable DNA polymerase. Recent studies have shown that certain thermostable DNA polymerases have the ability to reverse transcribe RNA, allowing the use of a single enzyme to amplify RNA amplicons (Myers and Gelfand (1991) Biochemistry 30:7661-7666). Since RNA is labile at high temperature in the presence of divalent ions, reverse transcription is carried out at lower temperature (50-60.degree. C.) than DNA synthesis. Therefore, it would be desirable to have a reagent that the reagent that is used to inhibit the ambient activity of the polymerase should reactivate the polymerase at lower temperature. This requirement eliminates the use of an antibody that demands high temperatures (70-90.degree. C.) for inactivation to generate in situ hot start conditions in RNA-based amplifications.